CN114292855B - PagARR9 gene for regulating and controlling growth of xylem of poplar and application thereof - Google Patents

Abstract

The invention discloses a PagARR9 gene for regulating and controlling the xylem development of poplar and application thereof, relating to the technical field of biological gene engineering; the nucleotide sequence and the amino acid sequence of the coding region of the PagARR9 gene are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4. According to the invention, the PagARR9 gene is transferred into the poplar, the xylem of the transgenic poplar which excessively expresses PagARR9 is obviously reduced compared with the wild type, the PagARR9 gene is knocked out through CRISPR/Cas9, and the xylem of the transgenic poplar is obviously increased compared with the wild type; the cytokinin response regulatory factor gene PagARR9 is a key regulatory factor for regulating and controlling the development of poplar xylem. The invention provides a new choice for a regulation and control means of xylem development, and has important application value in the field of forest genetic engineering.

Description

PagARR9 gene for regulating and controlling growth of xylem of poplar and application thereof

Technical Field

The invention relates to a PagARR9 gene for regulating and controlling the xylem development of poplar trees and application thereof, in particular to a cytokinin response regulatory factor PagARR9 gene for regulating and controlling the xylem development of poplar trees and application thereof, belonging to the technical field of plant genetic engineering.

Background

Modern forestry is responsible for maintaining the ecological safety of the state and the soil and meeting the important mission of strategic material supply of forest products such as wood requirements and the like. With the rapid development of economy, the consumption demand of China on forest resources and forest products, particularly wood, is continuously and greatly increased, and the wood is the second largest world import country. The improvement of the wood yield and the quality of the limited forestation land is very important by cultivating a new variety of fast-growing high-quality artificial forests. However, due to the limitations of the biological characteristics of trees, such as long growth period, high heterozygosity and the like, the genetic background of wood properties is rarely known so far, and the realization of the high-quality and high-yield targets of the artificial forest is restricted.

Therefore, the genetic regulation and control mechanism of wood formation is disclosed, the molecular biology theory of trees can be enriched, and support can be provided for the wood property improvement of trees by adopting a molecular breeding technology.

Forest cambium activity and secondary vascular tissue development are under the synergistic action of a plurality of external environmental factors and endogenous hormones, wherein cytokinin has important influence on the formation, division and differentiation of vascular cambium. The cambium activity and wood yield are obviously increased or reduced by increasing or reducing the cytokinin concentration in the poplar cambium formation layer area through regulating the cytokinin synthesis or metabolism key genes, but the molecular mechanism of regulating the cambium activity and the xylem development is not clear.

Cytokinin response regulatory factor RRs is a key factor in cytokinin signal transduction processes, and is the most abundant family of cytokinin-related regulatory genes, which regulate downstream signals through the regulation of transcriptional activity and protein activity.

In the arabidopsis genome, a total of 24 ARR gene families for coding all positive and negative cytokinin response regulating factors exist, functional redundancy exists among different members, and the ARR gene families are jointly involved in regulation and control of plant growth and development. Among them, the A-type ARR family proteins are mainly used as negative feedback regulation cytokinin signals, the B-type ARRs are plant specific transcription factors, and cytokinin signal pathways depend on the stability of the main B-type ARRs.

Unlike Arabidopsis, poplar as woody plant has self-renewing and persistent meristematic tissue, which can differentiate into terminal bud and flower bud to maintain perennial characteristic and unique meristem growth regulating mechanism. During the long-term evolution of plants, many important regulatory factors change the number of homologous gene families in perennial woody plants, and the gene functions may also be differentiated. 33 RRs family genes are found in poplar, different members have different expression modes, and different functions are performed in different biological processes.

Therefore, the method utilizes the poplar which is a model woody plant to research the molecular mechanism of the cytokinin response regulatory factor RR for regulating and controlling the wood formation and screen the specific ARR gene for regulating and controlling the cambium activity and the wood formation, and has important significance for understanding the cytokinin regulation cambium activity and the wood yield and the genetic engineering breeding of the poplar.

Disclosure of Invention

The invention aims to provide a gene PagARR9 capable of regulating and controlling the xylem development of a poplar, and an expression vector of the gene PagARR9 is used for transforming the poplar so as to promote the development of a forest molecular breeding technology, provide a technical means for the cultivation or screening of excellent tree species and lay a foundation for exploring the molecular mechanism of the xylem development.

In order to solve the technical problems, the invention adopts the following technical scheme:

based on the analysis and research on poplar cytokinin RRs family members, the invention identifies an expression gene PagARR9 capable of regulating and controlling the xylem development dominance of poplar, and the nucleotide sequence and the amino acid sequence of the coding region are respectively shown as SEQ ID No.3 and SEQ ID No. 4. The PagARR9 CDS has a total length of 600bp, and codes 199 amino acids and 1 stop codon.

Inserting xylem dominant expression gene PagARR9 behind strong promoter 35S to obtain an over-expression vector, and transforming poplar to obtain an over-expression plant.

And designing an sgRNA primer by using the conserved sequence, constructing a CRISPR/Cas9 vector of the PagARR9 gene, and transforming a poplar to obtain a PagARR9 knockout plant.

In the specific research process, the PagARR9 overexpression strains (OE-32, OE-37 and OE-38) and gene knockout plants (C9-2, C9-7 and C9-21) are respectively obtained by transforming Populus alba 84K (Populus alba multiplied by P. Glandulosa) by using the vector through an agrobacterium-mediated method.

The observation of phenotype and microscopic morphology of the obtained different transgenic strains shows that the PagARR9 overexpression strain is remarkably shortened, and xylem is remarkably reduced; while PagARR9 knockout lines were significantly elevated and xylem was significantly increased.

The research results prove that PagARR9 has a key regulation and control effect on the plant height and xylem development of poplar trees and has important application value in molecular breeding and fine variety breeding of forest trees.

Compared with the prior art, the invention has the main beneficial technical effects that:

according to the invention, the PagARR9 gene is screened and identified by taking silver adenophora poplar 84k as a material, and phenotype identification based on overexpression and knockout plants of the PagARR9 gene shows that the PagARR9 gene can regulate and control the xylem development of poplar, so that the PagARR9 gene is a key regulatory factor for regulating and controlling the xylem development of poplar, a new choice is provided for regulating and controlling means of the xylem development, and the PagARR9 gene has an important application value in the field of forest genetic engineering.

The present invention is further illustrated by the following detailed description and accompanying drawings, which are not meant to limit the scope of the invention.

Drawings

FIG. 1 is a diagram showing the analysis of the tissue expression profile of the PagARR9 gene, a cytokinin-responsive regulatory factor, in example 1 of the present invention.

FIG. 2 is a diagram showing the quantitative detection of the transcription level of the wild-type 84K poplar and the transgenic poplar overexpressing PagARR9 in example 1 of the present invention.

FIG. 3-1 shows the detection results of C9-2 knockout transgenic plants in example 1 of the present invention.

FIG. 3-2 shows the results of detection of C9-7 knockout transgenic plants in example 1 of the present invention.

FIGS. 3-3 show the results of detection of C9-21 knockout transgenic plants in example 1 of the present invention.

FIG. 4 is a diagram comparing the height phenotype of wild-type 84K poplar of example 1 of the present invention with that of transgenic poplar plants overexpressing PagARR9 and knockout gene.

FIG. 5-1 shows the statistics of wild-type 84K poplar and the stems of transgenic poplar with over-expressed PagARR9 and gene knockout in example 1 of the present invention.

FIG. 5-2 shows the statistics of plant heights of wild type 84K poplar and overexpressed PagARR9 and gene knocked-out transgenic poplar in example 1 of the present invention.

FIG. 6 is the observation of the woody tissue section of wild type 84K poplar and transgenic poplar overexpressing PagARR9 and gene knockout in example 1 of the present invention.

FIG. 7 shows the wood width statistics of wild-type 84K poplar and overexpressed PagARR9 and gene knocked-out transgenic poplar in example 1 of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples, wherein the operations not described in detail in the following examples can be realized by reference to the operations of molecular cloning and instructions for using the kit.

Unless otherwise specified, the reagents referred to in the following examples are commercially available conventional reagents, and the methods used are those commonly used in the art.

Example 1:

1. tissue expression analysis of poplar PagARR9 gene

1. Cloning of the PagARR9 Gene of Poplar

Extracting 84K poplar total RNA from silver glandular poplar 84K (P.alba X.glandulosa) by using an RNeasy Plant Mini kit and an RNase-free DNase I kit (Qiagen, hilden, germany); mu.g of RNA was taken for each sample, the first strand of cDNA was synthesized by using SuperScript III first-strand synthesis system (Life Technologies, carlsbad, calif., USA), the genomic sequence of Populus tomentosa was referenced, primers (amplicon containing start and stop codons) were designed using Primer3 software, and full-length amplification of the gene (GATEWAY linker was introduced in the primers) was performed.

Wherein, the PagARR9 ORF forward primer PagARR9-CDS-F is shown as SEQ ID NO.1 in the sequence table (Table 1), and the reverse primer PagARR9-CDS-R is shown as SEQ ID NO.2 (Table 2).

TABLE 1

TABLE 2

PCRThe reaction system is as follows: taKaRa high fidelity amplification enzyme mixture PrimeSTAR 12.5. Mu.l, forward primer (10. Mu.M) 1. Mu.l, reverse primer (10. Mu.M) 1. Mu.l, template (84K poplar cDNA) 1. Mu.l, sterile ddH ₂ O is complemented to 25 mu l; reaction procedure: pre-denaturation at 98 deg.C for 5min; 30s at 98 ℃;56 ℃ for 30s; at 72 ℃,3min,10 cycles; 30s at 98 ℃; 30s at 60 ℃; at 72 ℃,3min,25 cycles; 10min at 72 ℃; the finally obtained gene full-length cDNA sequence is 600bp, which is named as PagARR9 gene, the sequence is shown as a sequence table SEQ ID NO.3 (table 3), and the sequence of the compiled expression protein is shown as a sequence table SEQ ID NO.4 (table 4).

TABLE 3

TABLE 4

2. Analysis of tissue expression characteristics

Plant material was derived from individual tissues and organs of an 84K Yang Yesheng plant grown in the greenhouse for 2 months, including the following several parts, the apical meristem (SAM): comprises a first leaf and a second leaf which are not unfolded at the top end of the plant, and also comprises a stem section in the middle of the leaves; LEAF (LEAF): a continuous leaf from the 3 rd leaf to the 7 th leaf; BARK (BARK): bark from internode 15 to the base of the plant, this part of the tissue mainly covers the formation lamina area and phloem area; xylem (DIX): the stem section from the 15 th internode to the base of the plant, after the bark is removed, the developing xylem area is scraped; STEM Segment (STEM): all tissues between nodes 2-3, and between nodes 4, 6, 8, 10, 15, and 20, including the cortex, lamina-forming region, xylem region, and the like.

Each of the above samples contained three biological replicates, all plant material was frozen quickly with liquid nitrogen and stored in a-80 ℃ freezer for future use, sample RNA was extracted, cDNA was synthesized by reverse transcription, poplar ACTIN was used as an internal reference gene, and the tissue expression characteristics of the PagARR9 gene were analyzed by quantitative PCR using PagARR9-RT-F (SEQ ID No.5, table 5) and PagARR9-RT-R (SEQ ID No.6, table 6) as amplification primers.

TABLE 5

Name (R)	Sequence 5 (SEQ ID NO. 5)
		PagARR9-RT-F	GTGGAAATGGCTCTGGCTAC

TABLE 6

Name (R)	Sequence 6 (SEQ ID NO. 6)
		PagARR9-RT-R	TCAAGCCCAAAAACTCCAAG

The real-time fluorescent quantitative PCR reaction system is as follows (10. Mu.l reaction system):

the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; then 45 cycles of amplification were performed, comprising: denaturation at 95 ℃ for 20sec, annealing at 58 ℃ for 20sec, and extension at 72 ℃ for 20sec; finally, extending for 5min at 72 ℃; at the same time, the following process is adoptedObtaining a dissolution curve: in the process of heating from 60 ℃ to 95 ℃, the temperature is increased at a speed of 0.06 ℃/sec (5 acquistions per ℃), until the temperature is increased, the whole procedure is completed, and the operation is finished; by means of 2 ^-ΔΔCt And (4) carrying out construction fluorescence quantitative result analysis in a calculation mode.

FIG. 1 is a graph showing the analysis of the tissue expression profile of the PagARR9 gene, a cytokinin-responsive regulatory factor, in example 1 of the present invention; fluorescent quantitative PCR analysis shows that the PagARR9 gene has different expression levels in different tissues and relatively high expression abundance in roots, while the relative expression level is increased along with the increase of lignification degree of stem segments in stems, and the expression level of xylem is obviously higher than that of phloem, so that the gene is presumed to play an important role in the aspect of regulation and control of formation of secondary xylem.

2. Construction of plant expression vector for PagARR9 gene

1. Construction of overexpression vectors

Constructing an overexpression vector of the PagARR9 gene by using a GATEWAY technology, performing PCR amplification by using a specific PCR primer and 84K cDNA as a template, and constructing the ORF of the PagARR9 gene into an entry vector PDNOR222.1; the reaction system is 80ng of PCR product; PDNOR222.1 vector 0.4. Mu.l; BP enzyme (cat # invitrogen 11789020) 0.6. Mu.l; sterile ddH ₂ Make up to 5. Mu.l of O, the reaction program is: the reaction is carried out for 5h at 25 ℃.

Selecting positive clones from a screening culture plate for PCR detection and sequencing verification, after linearization of an entry vector with a PagARR9 gene by MluI restriction endonuclease, carrying out LR reaction with a plant expression vector PMDC32, wherein the reaction system is as follows: linearizing 50ng of PDNOR222.1 vector to which the PagARR9 gene has been ligated; PMDC32 vector 75ng; LR enzyme (cat # invitrogen 11791020) 0.6. Mu.l; make up to 5. Mu.l of water, reaction conditions: reacting for 5 hours at 25 ℃; after LR reaction, the PagARR9 gene is introduced into a plant expression vector PMDC32 to obtain an over-expression vector (PMDC 32-PagARR 9), and a strong expression promoter CaMV35S is assembled at the 5' end of the PagARR9 gene and can enable the PagARR9 gene to be efficiently expressed in a poplar body; the 3' end of the PagARR9 gene is assembled with a strong terminator NOS, which can effectively terminate the transcription of the PagARR9 gene.

2. Construction of Gene knockout vectors

(1) Designing a primer: the 84K poplar PagARR9 genome sequence is sourced, full-length genome primers are designed according to the genome sequence of the PtARR9 of Chinese white poplar, 84K poplar genome DNA is used as a template, the PagARR9 genome sequence is cloned, a 20bp random Nucleotide sequence (PAM) is selected at the position without SNP (Single Nucleotide Polymorphism) sites, a target primer sequence AtU dT1F (SEQ ID NO. 7) (Table 7), atU3dT1R (SEQ ID NO. 8) (Table 8), atU-1T 1F (SEQ ID NO. 9) (Table 9), atU6-29T1R (SEQ ID NO. 10) (Table 10) is synthesized.

TABLE 7

Name (R)	Sequence 7 (SEQ ID NO. 7)
		AtU3dT1F	gtcaCAGTGGAATTGTAATGGCTG

TABLE 8

Name(s)	Sequence 8 (SEQ ID NO. 8)
		AtU3dT1R	aaacCAGCCATTACAATTCCACTG

TABLE 9

Name (R)	SEQ ID NO. 9)
		AtU6-1T1F	gtcaTTCAAACCCAAAAACTCCA

Watch 10

Name (R)	SEQ ID NO. 10)
		AtU6-1T1R	aaacTGGAGTTTTTGGGTTTGAA

(2) Preparation of target sequence linkers: diluting the target sequence primers into 1 mu M mother solution by using TE Buffer solution or double distilled water, then respectively taking 10 mu l of upstream and downstream target sequence primers, uniformly mixing, performing 90 ℃ denaturation for 30s, then transferring to the room temperature condition, and gradually cooling and annealing to form double chains.

(3) A10. Mu.l portion of 1 XBsAI enzyme-cleaved ligation reaction solution (10. Mu.l portion) was prepared:

react for 5 cycles with a PCR instrument: 5min at 37 ℃;20 ℃ for 5min.

(4) The gRNA expression cassette was amplified using 2 rounds of PCR amplification to obtain more stable specific target products and to avoid amplification of the empty product.

1) Performing first round of PCR amplification, namely performing two PCR reaction amplifications aiming at a forward target sequence and a reverse complementary target sequence respectively, taking 1 mu l of enzyme digestion ligation product obtained in the step (3) as a template, and performing PCR reaction by using primers in tables 7, 8, 9 and 10, wherein the primer concentrations are 10 mu M:

the reaction conditions for both reaction 1 and reaction 2 were: 95 ℃ for 15sec;60 ℃ for 15sec;72 ℃,15sec; the cycle number is 25-28 cycles, 5 mul PCR product is taken to carry out 2% agarose gel electrophoresis detection; and taking 1. Mu.l of each PCR product of reaction 1 and reaction 2, and using ddH ₂ After 10-fold dilution with O, 2. Mu.l of the solution was aspirated as a template for the second PCR reaction.

TABLE 11

Name (R)	SEQ ID NO.11
		U-F	CTCCGTTTTACCTGTGGAATCG

2) Second round PCR reaction (50. Mu.l system):

the reaction conditions are the same as the first round of PCR reaction, 5 μ l of the obtained PCR product is taken and subjected to electrophoresis detection, and 45 μ l of the residual PCR product is purified and recycled by using a Takara purification kit to wait for the next enzyme digestion ligation reaction.

TABLE 12

Name (R)	SEQ ID NO. 12)
		B1’	TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG

Watch 13

Name(s)	Sequence 13 (SEQ ID NO. 13)
		BL	AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC

(5) pYLCRISPR/Cas9-DH final vector digestion ligation reaction (15. Mu.l system):

the reaction conditions are as follows: enzyme digestion is carried out for 2min at 37 ℃; the variable-temperature circulating enzyme cutting connection is about 10-15 cycles: 3min at 10 ℃; 5min at 20 ℃; finally, the enzyme digestion is carried out for 2min at 37 ℃.

(6) And (5) sucking the enzyme digestion ligation reaction solution in the step (5) to perform escherichia coli competent cell transformation to obtain pYLCRISPR/Cas9-DH vector plasmid containing the target sequence of ligation.

3. Genetic transformation and detection of PagARR9 gene

Genetic transformation of the PagARR9 Gene

The constructed over-expression vector (PMDC 32-PagARR 9) and gene knockout vector (pYLCIRPR/Cas 9-DH containing the target sequence of the connection purpose) are transferred into agrobacterium GV3101 by an electric shock method and transferred into poplar by agrobacterium-mediated genetic transformation, and the transformation steps are as follows: 84K poplar tissue culture seedlings for genetic transformation are cultured under the conditions that the culture temperature is 23-25 ℃, the illumination is 16/8h (day/night), the illumination intensity is 50 mu M m-2s-1, agrobacterium containing a target expression vector infects leaves when OD600= 0.6-0.8, the infected leaves are placed on an adventitious bud induction culture medium (SIM, murashige-Skoog (MS) basic culture medium, 0.5 mg/L6-benzylaminopurine (6-benzyl aminopurine) (6-BA) and 0.05mg/L Naphthalene Acetic Acid (NAA)) are added, and the mixture is cultured for 3 days under the dark condition that the temperature is 22 +/-2 ℃, transferring the leaves after co-culture to SIM containing 3mg/L hygromycin (hygromycin B) and 200mg/L Timentin, inducing and screening resistant adventitious buds under the conditions that the culture temperature is 23-25 ℃, the illumination is 16/8h (day/night) and the illumination intensity is 50 mu M m-2s-1, transferring the resistant adventitious buds to a rooting culture medium (RIM, 1/2MS basic culture medium is added with 0.05mg/L IBA and 0.02mg/L NAA) containing 3mg/L hygromycin (hygromycin B) and 200mg/L Timentin after 30 days of induction culture until rooting is induced, extracting rooted plant leaf DNA and performing PCR verification.

2. Detection of overexpressed transgenic plants

Obtaining a resistant PagARR9 gene overexpression 84K poplar and a wild plant, extracting genome DNA, amplifying the resistant gene on an expression vector by utilizing PCR (polymerase chain reaction), and obtaining a clear strip by amplification, namely a transgenic plant; selecting a stem segment of a transgenic plant, taking a wild-type stem segment as a control, extracting total RNA, carrying out reverse transcription, and carrying out quantitative analysis on a PagARR9 gene to determine the expression quantity of a target gene of the transgenic plant, wherein quantitative primers are PagARR9-RT-F (SEQ ID NO. 5) and PagARR9-RT-R (SEQ ID NO. 6).

As shown in FIG. 2, a graph is shown for quantitative detection of transcription levels of wild-type 84K poplar and transgenic poplar over-expressing PagARR9 in example 1, wherein the expression levels of PagARR9 gene in over-expressing transgenic lines OE-32, OE-37 and OE-38 are 14.4, 12.9 and 12.3 times of that in wild-type 84K, respectively.

CRISPR-Cas 9-mediated PagARR9 gene editing mutant plant identification

Designing mutant plant detection primers 9-C9-F617 (SEQ ID NO.14, table 14) and 9-C9-R617 (SEQ ID NO.15, table 15), wherein the target band comprises the PAM position of the PagARR9 gene; extracting transgenic plant DNA as a template, performing PCR amplification by using high fidelity enzyme (Prime STARMax DNA polymerase), connecting an obtained PCR product to a T vector (Idela zero background pTOPO-TA/Blunt universal cloning kit, idela, beijing) and converting the PCR product into a large intestine susceptible cell, identifying the PCR product as a positive clone by using bacterial liquid, sending the positive clone to a sequencing company for sequencing, selecting more than 30 monoclonals for sequence comparison analysis, and obtaining sequencing results as shown in figures 3-1 to 3-3, wherein the sequencing results are shown in figure 3-1, the detection result is a C9-2 knockout transgenic plant detection result in the embodiment 1 of the invention, and the sequencing result shows that 274 bases (sequences in a table 16) on two chromosomes of the plant are replaced by 275 bases (sequences in a table 17) which are coded differently; as shown in FIG. 3-2, the test result of the C9-7 knockout transgenic plant in example 1 of the present invention shows that the editing mode of target gene sequence deletion of 1 base exists on both chromosomes of the strain according to the sequencing result; as shown in FIGS. 3-3, the results of the detection of the C9-21 knockout transgenic plant in example 1 of the present invention; as can be seen from the sequencing results, there is an editing mode of target gene sequence deletion of 2 bases on both chromosomes in the strain.

TABLE 14

Name (R)	Sequence 14 (SEQ ID NO. 14)
		9-C9-F617	AGTCCCTCACCTTCCCTTCTTTC

Watch 15

Name (R)	Sequence 15 (SEQ ID NO. 15)
		9-C9-R617	GCCACATAGTCAGATTTCTAGCATA

TABLE 16

TABLE 17

4. PagARR9 transgenic plant phenotype observation

The PagARR9 mutant plant, the overexpression plant and the wild-type 84K poplar are used as controls, the plants are planted in a greenhouse at the same time, three batches of plants are planted, phenotype determination and photographing are respectively carried out on the plants in different batches, as shown in figure 4, a comparison graph of the high phenotype of the wild-type 84K poplar, the overexpression PagARR9 and the transgenic poplar with gene knockout in the embodiment 1 is shown, figure 4 shows a phenotype photo after transplanting for two months, compared with the non-transgenic 84K Yang Zhizhu, the mutant plant is high and thick as a whole, and the overexpression plant shows a delicate and small phenotype; as shown in FIG. 5-1, statistics of wild type 84K poplar and transgenic poplar stem overexpressing PagARR9 and gene knockout in example 1 of the present invention are shown; as shown in FIG. 5-2, statistics of the plant heights of the wild-type 84K poplar and the transgenic poplar with over-expressed PagARR9 and gene knockout in example 1 of the present invention are shown; on the plant height, the mutant C9-21 plants are remarkably increased by 24 percent, and the C9-7 and C9-2 plants are remarkably increased by 12 percent and 14 percent respectively; the height reduction of the over-expressed plants also reaches a very significant level, and the heights of OE-32, OE-37 and OE-38 are respectively reduced by 20%, 22% and 24%, and the results are shown in FIG. 5-1; on the change of the thickness of the ground diameter, the C9-21, C9-7 and C9-2 plants are respectively thickened by 31%, 27% and 25% to reach the extremely significant level, the over-expressed plant OE-32 is thinned by 12.5% to reach the extremely significant level, and both OE-37 and OE-38 are thinned by 6% to reach the significant level, and the results are shown in figure 5-2. Therefore, combining phenotype and statistical data, it was preliminarily presumed that PagARR9 has a function of inhibiting division, proliferation and differentiation of stem cell segments.

5. PagARR9 transgenic xylem section observations

1. Taking a stem section 2cm above the ground of a soil-cultured seedling poplar with the length of 0.5cm, fixing the stem section in a Leica VT1200S slicing groove of a vibration slicing machine by LOCTITE 495 glue, cutting the stem section into cross section slices with the thickness of 50 mu m, and storing the slices in 70% alcohol.

2. Histochemical staining and photographic observation analysis: after the fresh sections are subjected to TBO staining for 1min at 0.05 percent, the sections are washed with water for three times to remove floating color and redundant staining solution, a cover glass is covered, an Olympus BX51 common optical microscope is adopted to observe the sections subjected to TBO staining, pictures are taken to analyze the morphological change of the cross section of the stem section, and the result is shown in figure 6 and is observed by the wood tissue sections of the wild 84K poplar and the transgenic poplar which excessively expresses PagARR9 and is subjected to gene knockout in the embodiment 1 of the invention; the widening and diameter thickening phenomena of the xylem region of the PagARR9 mutant plants compared to 84K Yang Duizhao; in contrast, overexpressing transgenic plants show xylem narrowing and thinning; the xylem widths of the C9-21 and C9-2 plants were counted and found to be increased by 45% and 50%, respectively, while those of OE-32 and OE-38 plants were decreased by 25% and 12%, respectively, and the results are shown in FIG. 7, which is a statistics of the xylem widths of the wild type 84K poplar and the transgenic poplar with overexpressed PagARR9 and gene knockout in example 1 of the present invention. These results above demonstrate that the PagARR9 gene influences the development of secondary xylem, i.e.radial growth.

The phenotype and microscopic morphology observation of the obtained different transgenic strains shows that the heights of the PagARR9 overexpression strains are obviously shortened, and xylem is obviously reduced; and the PagARR9 knockout strain has obviously increased plant height and xylem. The research results prove that the PagARR9 has a key regulation and control effect on the plant height and xylem development of the poplar, and has important application values in molecular breeding and fine variety breeding of forest trees.

Although the invention has been described in detail with respect to the preferred embodiments and examples, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the appended claims.

Sequence listing

<110> forestry research institute of China forestry science research institute

<120> PagARR9 gene for regulating and controlling poplar xylem development and application thereof

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 48

<212> DNA

<213> Artificial sequence (PagARR 9-CDS-F)

<400> 1

ggggacaact ttgtacaaaa aagttggaat ggctgtggaa atggctct 48

<210> 2

<211> 55

<212> DNA

<213> Artificial sequence (PagARR 9-CDS-R)

<400> 2

ggcggccgca caactttgta caagaaagtt gggtatcaga caacttccaa gccat 55

<210> 3

<211> 600

<212> DNA

<213> Artificial sequence (PagARR 9)

<400> 3

atggctgtgg aaatggctct ggctactacc atggctactg agactcagtt tcatgttctt 60

gctgttgacg actgccttat tgacagaaag ttgattgaaa ggctccttaa aacctcttct 120

tatcaagtca cggcagtgga ttcaggaagc aaggccttgg agtttttggg tttgaatgga 180

gaaaatgagc tgagagattc aaaacctgcc tctgtttccc ctgaccccta tcaccagcac 240

cttgaaatta atatgatcat tacagattac tgtatgccag gaatgacagg ctatgatctt 300

ctaaaaaaga tcaaggaatc taaatatttc aaggacatcc cagttgtgat catgtcctca 360

gagaatgttc catcaagaat caacagatgc ctaaaagaag gagctgaaga gttcttcttg 420

aagccggttc aattatcaga tgtcaacaag cttagacccc atctaatgaa gggaagatgc 480

aaggaagaag aagaagaaga agaagatcaa cccaataaca agagaaaggg catggaagaa 540

attgttaact ctccagatcg aacaagaaca agatacaatg atggcttgga agttgtctga 600

<210> 4

<211> 199

<212> PRT

<213> Artificial sequence (PagARR 9)

<400> 4

Met Ala Val Glu Met Ala Leu Ala Thr Thr Met Ala Thr Glu Thr Gln

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Phe His Val Leu Ala Val Asp Asp Cys Leu Ile Asp Arg Lys Leu Ile

20 25 30

Glu Arg Leu Leu Lys Thr Ser Ser Tyr Gln Val Thr Ala Val Asp Ser

35 40 45

Gly Ser Lys Ala Leu Glu Phe Leu Gly Leu Asn Gly Glu Asn Glu Leu

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Arg Asp Ser Lys Pro Ala Ser Val Ser Pro Asp Pro Tyr His Gln His

65 70 75 80

Leu Glu Ile Asn Met Ile Ile Thr Asp Tyr Cys Met Pro Gly Met Thr

85 90 95

Gly Tyr Asp Leu Leu Lys Lys Ile Lys Glu Ser Lys Tyr Phe Lys Asp

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Ile Pro Val Val Ile Met Ser Ser Glu Asn Val Pro Ser Arg Ile Asn

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Arg Cys Leu Lys Glu Gly Ala Glu Glu Phe Phe Leu Lys Pro Val Gln

130 135 140

Leu Ser Asp Val Asn Lys Leu Arg Pro His Leu Met Lys Gly Arg Cys

145 150 155 160

Lys Glu Glu Glu Glu Glu Glu Glu Asp Gln Pro Asn Asn Lys Arg Lys

165 170 175

Gly Met Glu Glu Ile Val Asn Ser Pro Asp Arg Thr Arg Thr Arg Tyr

180 185 190

Asn Asp Gly Leu Glu Val Val

195

<210> 5

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<212> DNA

<213> Artificial sequence (PagARR 9-RT-F)

<400> 5

gtggaaatgg ctctggctac 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (PagARR 9-RT-R)

<400> 6

tcaagcccaa aaactccaag 20

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (AtU dT 1F)

<400> 7

gtcacagtgg aattgtaatg gctg 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (AtU dT 1R)

<400> 8

aaaccagcca ttacaattcc actg 24

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence (AtU-1T 1F)

<400> 9

gtcattcaaa cccaaaaact cca 23

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (AtU-1T 1R)

<400> 10

aaactggagt ttttgggttt gaa 23

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence (U-F)

<400> 11

ctccgtttta cctgtggaat cg 22

<210> 12

<211> 40

<212> DNA

<213> Artificial sequence (B1')

<400> 12

ttcagaggtc tctctcgact agtggaatcg gcagcaaagg 40

<210> 13

<211> 40

<212> DNA

<213> Artificial sequence (BL)

<400> 13

agcgtgggtc tcgaccgacg cgtccatcca ctccaagctc 40

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence (9-C9-F617)

<400> 14

agtccctcac cttcccttct ttc 23

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (9-C9-R617)

<400> 15

gccacatagt cagatttcta gcata 25

<210> 16

<211> 274

<212> DNA

<213> Artificial sequence (C9-2-Before edge)

<400> 16

ctgtggaaat ggctctggct actaccatgg ctactgagac tcagtttcat gttcttgctg 60

ttgacgactg ccttattgac agaaagttga ttgaaaggct ccttaaaacc tcctcttatc 120

aaggtacaac cacagccatg tacatggatg tcgaagcccc aagggtttct tggattatat 180

attattttga tcttggattc ttgattgctg acaatttttt tggttttggg tcatttttac 240

agtcacggca gtggattcag gaagcaaggc cttg 274

<210> 17

<211> 275

<212> DNA

<213> Artificial sequence (C9-2-After-edited)

<400> 17

tccaaggcct tgcttcctga atccactgcc gtgactgtaa aaatgaccca aaaccaaaaa 60

aattgtcagc aatcaagaat ccaagatcaa aataatatat aatccaagaa acccttgggg 120

cttcgacatc catgtacatg gctgtggttg taccttgata agaggaggtt ttaaggagcc 180

tttcaatcaa ctttctgtca ataaggcagt cgtcaacagc aagaacatga aactgagtct 240

cagtagccat ggtagtagcc agagccattt ccaca 275

Claims (1)

1. The application of the PagARR9 gene in regulating and controlling the development process of the xylem of the poplar is characterized in that: the nucleotide sequence and the amino acid sequence of the coding region of the PagARR9 gene are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4; the regulation and control of the growth process of the xylem of the poplar refers to narrowing and thinning the xylem region of the poplar plant when the PagARR9 gene is overexpressed; when the PagARR9 gene is knocked out, the xylem region of the poplar plant will be widened and thickened in diameter.

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